Cryptosporidiosis, a widespread diarrheal disease caused by Cryptosporidium (CP) species infection, is an emerging global health problem associated with high morbidity and mortality. The Global Enteric Multicenter Study (GEMS) recently reported this neglected protozoan parasite as 1 of the 4 major diarrheal pathogens worldwide. Although previously known for causing chronic diarrhea in AIDS patients and for major waterborne outbreaks in the Western world, cryptosporidiosis has garnered medical and scientific attention only recently with appreciation of an urgent need to develop effective therapeutic strategies. The current treatment options are severely limited with no vaccines to date and the only FDA approved drug nitazoxanide exhibits limited efficacy. The parasite enters human host via fecal-oral route and infects the host intestinal epithelial cells. A major hurdle in drug development is the poor knowledge of host-parasite interactions primarily due to the lack of a physiologically relevant disease model recapitulating the native human intestinal in vivo environment of the parasite. Animal models are suboptimal for human infection studies, whereas human intestinal transformed cell lines do not truly represent host-parasite interactions in vivo. In this regard, a recent technology to generate human enteroids, small intestinal crypt-derived 3-D organoids with an epithelial layer surrounding a lumen, provided an ex-vivo model truly recapitulating the architecture and functional diversity of the native epithelium. Human enteroids and enteroid-derived polarized monolayers grown on Transwell inserts with distinct apical/basolateral cell surfaces provide exceptional opportunity as physiologically relevant model of human intestine to study host-pathogen interactions. Therefore, we propose the current exploratory studies to establish an ex vivo model of cryptosporidiosis utilizing crypt-derived human enteroids and enteroid-derived monolayers to investigate host-parasite interactions relevant to diarrheal diseases. Diarrhea mainly results from dysregulated intestinal ion and fluid transport (due to decreased absorption and/or increased secretion) and may also involve disruption of intestinal barrier function. Therefore, we hypothesized that a human enteroid model of CP infection will allow, for the first time, characterization of CP-induced dysregulation of epithelial ion transport and barrier function in the human intestine and define novel targets for intervention. The Specific Aims include: 1) Characterize invasion of C. parvum in human 3-D enteroids/enteroid-derived 2-D monolayers and the effects on epithelial barrier structure and function; 2) Elucidate mechanisms of altered ion transport and luminal fluid accumulation following C. parvum infection of human enteroids/enteroid-derived monolayers. This novel ex-vivo model of cryptosporidiosis should help overcome a major hurdle in studying host-parasite interactions relevant to human infection, identify superior therapeutic targets and establish a highly effective pre-clinical model for high throughput drug screening and thereby forming the basis for a future robust program in drug discovery and development.